The search for sensitive urinary biomarkers of kidney performance has attracted important efforts due to their potential value as clinical tools for detecting early signs of various diseases. The recent burst of powerful proteomic techniques such as ESi-MS, DESI-MS, MALDI-TOF-MS, SELDI-TOF-MS, DIOS-MS or LC-MS that allow sample screening across a statistically meaningful patient population, has led to the discovery and identification of distinctive urinary biomarkers of kidney dysfunction (Dihazi et al., Expert Rev Proteomics. 2007 February; 4(1):39-50)
In clinical nephrology, successful management of transplantation patients requires early detection and implementation of the most appropriate therapy. Available diagnostic methods include biochemical parameters and biopsies but the only non-invasive biomarker of allograft dysfunction is the insensitive, non-specific creatinine which does not allow detection of fibrosing changes at an early stage.
Research efforts have focused on urinary biomarkers of disease. A classical urinary proteome study reported that an unbiased proteomic approach can detect urine protein peaks associated with acute tubulointerstitial renal allograft rejection. Identification of these protein peaks by mass spectrometry demonstrated that they all derive from cleaved forms of beta2-microglobulin, presumably reflective of damaged renal proximal tubule cells (Schaub et al., 2005. Am. J. Transplant. 5, 729-738). Many studies followed this trend, to name a few: Dihazi et Al., Clin Chem. 2007 September; 53(9):1636-45; O'valle et Al., Transplant Proc. 2007 September; 39(7):2099-101; Peng et al., J Int Med Res. 2007 July-August; 35(4):442-9; WO-07121922-A2 and WO-07104537-A2). The number of published urinary biomarker studies is increasing rapidly. Most studies have looked at the soluble urine protein fraction, focusing on the identification of potential biomarkers in renal disease and diseases of the urogenital tract. They include studies of acute kidney injury, acute renal allograft rejection, glomerular disease and carcinoma of the kidney, bladder and prostate. In many cases the reported biomarkers remain unidentified, although some studies have identified a few biomarker proteins (O'Riordan et al., Am J Transplant 2007; 7:930-40; Schaub et al., 2005. Am. J. Transplant. 5, 729-738).
Notwithstanding these efforts, at the moment, standards for healthy renal function have not been the subject of any study. Traditionally, healthy renal function is assumed when a patient shows no signs of disease or pain. Monitoring healthy renal function may help identifying disorders at an early stage but, most importantly, it would provide clinicians with a powerful tool for managing patients with a grafted kidney.
A few urinary proteins that are present in samples of individuals with healthy kidney function but undergo a sharp reduction in disease, are known. The most prominent example is the Tamm-Horsfall glycoprotein (TH protein), also known as uromodulin (Kidney International, Vol. 16 (1979), pp. 279-289; JBC Vol. 265, No. 34, December 5, pp. 20764-20789, 1990). The TH protein is generally the most abundant protein in urine, and its presence can interfere with the detection of other proteins if it is not removed (Pisitkun et al., 2004. Proc. Natl. Acad. Sci. U.S.A. 101, 13368-13373). It can be depolymerised by use of reducing agents and warming. Human TH protein forms high molecular weight aggregates and may be dissociated into smaller molecular weight subunits of approximately 100 kDa by several agents (Stevenson et al., Biochem J 116:791-796, 1970). Thus, it is a common use in the field of urinary biomarker discovery to free the urine samples from uromodulin because it has no attributed diagnosing value. Uromodulin has, however, attracted certain interest because of its anti-inflammatory properties. The Japanese patent application JP-2002125673-A discloses a new uromodulin human gene and its uses as anti-inflammatory agent.
Other abundant urinary proteins are kinins. Mindroiu et al. (J Biol Chem. 1986 Jun. 5; 261(16):7407-11) compared the types of kinins excreted in fresh urine of dogs, rats, and humans using high performance liquid chromatography. They found that in human urine, the content of kinins was roughly 12% bradykinin, 30% Lys-bradykinin, 2% des-Arg1-bradykinin, and 41% of a then unknown kinin which was identical to Lys-Bradykinin but had an alanine in position 4 instead of a proline. Human urinary kallikrein incubated with semipurified human low molecular weight kininogen released 76% of the total kinins as Lys-bradykinin, 7% as bradykinin, and 17% as [Ala3]Lys-bradykinin. Thus lys-bradykinin is the most abundant type of kinin in urine, however, and despite its application in cardiovascular pathophysiology, no diagnostic value for renal pathophysiology has derived from its presence in urine.
A standard that positively asserts the fitness of a grafted kidney function would be of great value for clinicians in detecting early, otherwise undetectable, fibrosing changes.
Taking advantage of novel LC-MS techniques and owing to their innovative strategy for urinary biomarker discovery, the inventors have identified novel natural urinary peptides. All of them show reduced urinary concentrations in disease which qualify them as biomarkers of healthy kidney performance. Therefore, the present invention provides the physician with a clinical tool for diagnosing, instead of assuming, healthy renal physiology.